A wide variety of microorganisms will be screened for the presence of Type II restriction endonucleases. All such enzymes found will be characterized as to their recognition properties. This will include the determination of the sequence recognized and the precise site of cleavage. In those cases where the new enzyme shows a specificity identical with some previously discovered enzyme, the new enzyme will be tested for its ability to cleave DNA modified against the action of the old enzyme. Specific attention will be paid to methods that allow the discovery of enzymes with recognition sequences longer than 6 nucleotides, since such enzymes, which cleave DNA infrequently, are of great intrinsic value for mapping large genomes such as the human genome. An important aspect of this work is the preparation and maintenance of a cohesive and comprehensive database of information about restriction endonucleases. Procedures will be developed for the reliable dissemination of that database electronically to the BIONET computer resource and to other off- site locations. Procedures will be developed for the automatic updating of information in the database. In addition, a comprehensive package of programs will be developed to assist in the determination of new restriction enzyme recognition sites. This package will allow the entry of data about fragment lengths, numbers of sites, and approximate mapping information so that accurate predictions of restriction enzyme recognition sites can be made. Such predictions will then be tested experimentally. A major goal is to make available a wide variety of new restriction enzymes with novel specificities for use by the research community. Both the large collection of bacterial strains containing restriction enzymes and the major database of information about restriction enzymes that has been compiled at Cold Spring Harbor Laboratory will be maintained and made available to the scientific community. While this work will impact all areas of molecular biology, it is of special importance for chromosome mapping and genetic engineering.